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Patrick R Maloney,
Pasha Khan,
Michael Hedrick,
Palak Gosalia,
Monika Milewski,
Linda Li,
Gregory P Roth,
Eduard Sergienko,
Eigo Suyama,
Eliot Sugarman, [......],
Ying Su,
Derek Stonich,
Hung Nguyen,
Fu-Yue Zeng,
Arianna Mangravita Novo,
Michael Vicchiarelli,
Jena Diwan,
Thomas D Y Chung,
Layton H Smith,
Anthony B Pinkerton
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ABSTRACT: The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ∼330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.
Bioorganic & medicinal chemistry letters 09/2012; 22(21):6656-60. · 2.65 Impact Factor
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Janina Preuss,
Patrick Maloney,
Satyamaheshwar Peddibhotla,
Michael P Hedrick,
Paul Hershberger,
Palak Gosalia,
Monika Milewski,
Yujie Linda Li,
Eliot Sugarman,
Becky Hood, [......],
Danielle McAnally,
Layton H Smith,
Gregory P Roth,
Jena Diwan,
Thomas D Y Chung,
Esther Jortzik,
Stefan Rahlfs,
Katja Becker,
Anthony B Pinkerton,
Lars Bode
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ABSTRACT: A high-throughput screen of the NIH's MLSMR collection of ∼340000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is important for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In P. falciparum , the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 11 (ML276), is a submicromolar inhibitor of PfG6PD (IC(50) = 889 nM). It is completely selective for the enzyme's human isoform, displays micromolar potency (IC(50) = 2.6 μM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting PfG6PD in vivo are in progress.
Journal of Medicinal Chemistry 07/2012; 55(16):7262-72. · 4.80 Impact Factor
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Torkel Vang,
Wallace H Liu,
Laurence Delacroix,
Shuangding Wu, Stefan Vasile,
Russell Dahl,
Li Yang,
Lucia Musumeci,
Dana Francis,
Johannes Landskron,
Kjetil Tasken,
Michel L Tremblay,
Benedicte A Lie,
Rebecca Page,
Tomas Mustelin,
Souad Rahmouni,
Robert C Rickert,
Lutz Tautz
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Torkel Vang,
Wallace H Liu,
Laurence Delacroix,
Shuangding Wu, Stefan Vasile,
Russell Dahl,
Li Yang,
Lucia Musumeci,
Dana Francis,
Johannes Landskron,
Kjetil Tasken,
Michel L Tremblay,
Benedicte A Lie,
Rebecca Page,
Tomas Mustelin,
Souad Rahmouni,
Robert C Rickert,
Lutz Tautz
[show abstract]
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ABSTRACT: Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T-cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here we studied the spatiotemporal dynamics of the LYP-CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it downmodulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T-cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single-nucleotide polymorphism that confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a mutant LYP that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity.
Nature Chemical Biology 03/2012; 8(5):437-46. · 14.69 Impact Factor
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Charitha Madiraju,
Kate Welsh,
Michael P Cuddy,
Paulo H Godoi,
Ian Pass,
Tram Ngo, Stefan Vasile,
Eduard A Sergienko,
Paul Diaz,
Shu-Ichi Matsuzawa,
John C Reed
[show abstract]
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ABSTRACT: UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.
Journal of Biomolecular Screening 02/2012; 17(2):163-76. · 2.05 Impact Factor
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Eduard Sergienko,
Jian Xu,
Wallace H Liu,
Russell Dahl,
David A Critton,
Ying Su,
Brock T Brown,
Xochella Chan,
Li Yang,
Ekaterina V Bobkova, [......],
Hongbin Yuan,
Justin Rascon,
Sharon Colayco,
Shyama Sidique,
Nicholas D P Cosford,
Thomas D Y Chung,
Tomas Mustelin,
Rebecca Page,
Paul J Lombroso,
Lutz Tautz
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ABSTRACT: The hematopoietic protein tyrosine phosphatase (HePTP) is implicated in the development of blood cancers through its ability to negatively regulate the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Small-molecule modulators of HePTP activity may become valuable in treating hematopoietic malignancies such as T cell acute lymphoblastic leukemia (T-ALL) and acute myelogenous leukemia (AML). Moreover, such compounds will further elucidate the regulation of MAPKs in hematopoietic cells. Although transient activation of MAPKs is crucial for growth and proliferation, prolonged activation of these important signaling molecules induces differentiation, cell cycle arrest, cell senescence, and apoptosis. Specific HePTP inhibitors may promote the latter and thereby may halt the growth of cancer cells. Here, we report the development of a small molecule that augments ERK1/2 and p38 activation in human T cells, specifically by inhibiting HePTP. Structure-activity relationship analysis, in silico docking studies, and mutagenesis experiments reveal how the inhibitor achieves selectivity for HePTP over related phosphatases by interacting with unique amino acid residues in the periphery of the highly conserved catalytic pocket. Importantly, we utilize this compound to show that pharmacological inhibition of HePTP not only augments but also prolongs activation of ERK1/2 and, especially, p38. Moreover, we present similar effects in leukocytes from mice intraperitoneally injected with the inhibitor at doses as low as 3 mg/kg. Our results warrant future studies with this probe compound that may establish HePTP as a new drug target for acute leukemic conditions.
ACS Chemical Biology 11/2011; 7(2):367-77. · 6.45 Impact Factor
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Pasha M Khan,
Ricardo G Correa,
Daniela B Divlianska,
Satyamaheshwar Peddibhotla,
E Hampton Sessions,
Gavin Magnuson,
Brock Brown,
Eigo Suyama,
Hongbin Yuan,
Arianna Mangravita-Novo,
Michael Vicchiarelli,
Ying Su, Stefan Vasile,
Layton H Smith,
Paul W Diaz,
John C Reed,
Gregory P Roth
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ABSTRACT: NOD1 (nucleotide-binding oligomerization domain 1) protein is a member of the NLR (NACHT and leucine rich repeat domain containing proteins) protein family, which plays a key role in innate immunity as a sensor of specific microbial components derived from bacterial peptidoglycans and induction of inflammatory responses. Mutations in NOD proteins have been associated with various inflammatory diseases that affect NF-κB (nuclear factor κB) activity, a major signaling pathway involved in apoptosis, inflammation, and immune response. A luciferase-based reporter gene assay was utilized in a high-throughput screening program conducted under the NIH-sponsored Molecular Libraries Probe Production Center Network program to identify the active scaffolds. Herein, we report the chemical synthesis, structure-activity relationship studies, downstream counterscreens, secondary assay data, and pharmacological profiling of the 2-aminobenzimidazole lead (compound 1c, ML130) as a potent and selective inhibitor of NOD1-induced NF-κB activation.
ACS Medicinal Chemistry Letters 10/2011; 2(10):780-785. · 3.36 Impact Factor
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Ekaterina V Bobkova,
Wallace H Liu,
Sharon Colayco,
Justin Rascon, Stefan Vasile,
Carlton Gasior,
David A Critton,
Xochella Chan,
Russell Dahl,
Ying Su,
Eduard Sergienko,
Thomas D Y Chung,
Tomas Mustelin,
Rebecca Page,
Lutz Tautz
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ABSTRACT: Protein tyrosine phosphatases (PTPs) have only recently become the focus of attention in the search for novel drug targets despite the fact that they play vital roles in numerous cellular processes and are implicated in many human diseases. The hematopoietic protein tyrosine phosphatase (HePTP) is often found dysregulated in preleukemic myelodysplastic syndrome (MDS), as well as in acute myelogenous leukemia (AML). Physiological substrates of HePTP include the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Specific modulators of HePTP catalytic activity will be useful for elucidating mechanisms of MAPK regulation in hematopietic cells, and may also provide treatments for hematopoietic malignancies such as AML. Here we report the discovery of phenoxyacetic acids as inhibitors of HePTP. Structure-activity relationship (SAR) analysis and in silico docking studies reveal the molecular basis of HePTP inhibition by these compounds. We also show that these compounds are able to penetrate cell membranes and inhibit HePTP in human T lymphocytes.
ACS Medicinal Chemistry Letters 02/2011; 2(2):113-118. · 3.36 Impact Factor
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Ekaterina V Bobkova,
Wallace H Liu,
Sharon Colayco,
Justin Rascon, Stefan Vasile,
Carlton Gasior,
David A Critton,
Xochella Chan,
Russell Dahl,
Ying Su,
Eduard Sergienko,
Thomas D Y Chung,
Tomas Mustelin,
Rebecca Page,
Lutz Tautz
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ABSTRACT: Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high-throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique.
Biochemical and Biophysical Research Communications 02/2010; 394(1):194-9. · 2.48 Impact Factor
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Lutz Tautz,
Tomas Mustelin,
Shuangding Wu,
Sofie Vossius,
Souad Rahmouni, Stefan Vasile,
Eduard Sergienko,
Derek Stonich,
Hongbin Yuan,
Ying Su,
Russell Dahl,
Yalda Mostofi,
Thomas DY Chung
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ABSTRACT: Vaccinia H1-related (VHR) protein tyrosine phosphatase dephosphorylates and thereby inactivates extracellular signal-regulated kinases Erk1/2 and c-Jun N-terminal kinases Jnk1/2. These mitogen-activated protein (MAP) kinases mediate major signaling pathways triggered by extracellular growth factor, stress, or cytokines and regulate cellular processes such as differentiation, proliferation and apoptosis. Unlike many MAP kinase phosphatases (MKPs), VHR expression is not induced in response to activation of MAP kinases, but is instead regulated during cell cycle progression. The loss of VHR causes cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells without detrimental effects on normal cells. Here we report the development of multidentate small-molecule inhibitors of VHR that inhibit its enzymatic activity at nanomolar concentrations and are selective for VHR over HePTP and MKP-1. This novel small molecular probe, ML113 (CID-6161281) appears to interact with both the phosphate-binding pocket and several distinct hydrophobic regions within VHR's active site. As a result, it will serve as a useful tool in probing these interactions and elucidating the molecular mechanism underlying the selectivity against this phosphatase, in addition to providing greater understanding of the functional consequences for cancer biology.
01/2010;
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Eduard Sergienko,
Ekatarina Bobkova, Stefan Vasile,
Hongbin Yuan Ying Su,
Russell Dahl,
Li Yang,
Thomas DY Chung,
Shakeela Dad,
Tomas Mustelin,
Wallace Liu,
David A Critton,
Rebecca Page,
Lutz Tautz
[show abstract]
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ABSTRACT: Tyrosine phosphorylation plays a key role in signal transduction and the regulation of a broad set of physiological processes characteristic of multicellular organisms. Hematopoietic protein tyrosine phosphate (HePTP) is a tyrosine phosphatase expressed in hematopoietic cells with specificity for the dephosphorylation of Erk and p38 MAP kinases (MAPKs). It has been found that HePTP is often dysregulated in the preleukemic disorder myelodysplastic syndrome, as well as in acute myelogeneous leukemia. The identified probe ML119 (CID-1357397) selectively inhibits HePTP activity. Since this probe scaffold was discovered through its activity in the HePTP assays with small molecule substrates, it is proposed that this inhibition occurs through direct targeting of the HePTP active site. The probe inhibits through fast equilibrium as judged by the lack of apparent effect when pre-incubated with phosphatase proteins. Thus, small molecule inhibitors of HePTP would be useful as molecular probes for studying the mechanism of signal transduction and MAP kinase regulation. In addition, these probes may have therapeutic potential for the treatment of hematopoietic malignancies, such as acute myeloid leukemia, where HePTP has been reported to be overexpressed.
01/2010;
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Eduard Sergienko,
Ekaterina Bobkova,
Sharon Colayco,
Justin Rascon,
Xochella Chan, Stefan Vasile,
Ying Su,
Russell Dahl,
Thomas DY Chung,
Shakeela Dad,
Tomas Mustelin,
Wallace Liu,
David A Critton,
Rebecca Page,
Lutz Tautz
[show abstract]
[hide abstract]
ABSTRACT: Tyrosine phosphorylation is a key mechanism for signal transduction and the regulation of a broad set of physiological processes characteristic of multicellular organisms. Hematopoietic protein tyrosine phosphate (HePTP) is a tyrosine phosphatase expressed in hematopoietic cells with specificity for the dephosphorylation of Erk and p38 MAP kinases (MAPKs). It has been found that HePTP is often dysregulated in the preleukemic disorder myelodysplastic syndrome, as well as in acute myelogeneous leukemia. The identified probe ML120 (CID-4302116) selectively inhibits HePTP activity. Since this probe scaffold was discovered through its activity in the HePTP assays with small molecule substrates, it is proposed that this inhibition occurs through direct targeting of the HePTP active site. No time-dependent inhibition is observed as demonstrated by the linear progress curves of the HePTP phosphatase reaction in the presence of various concentrations of the ML120. Thus, small molecule inhibitors of HePTP would be useful as molecular probes for studying the mechanism of signal transduction and MAP kinase regulation. In addition, these probes may have therapeutic potential for the treatment of hematopoietic malignancies, such as acute myeloid leukemia, where HePTP has been reported to be overexpressed.
01/2010;
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Shuangding Wu,
Sofie Vossius,
Souad Rahmouni,
Ana V Miletic,
Torkel Vang,
Jesus Vazquez-Rodriguez,
Fabio Cerignoli,
Yutaka Arimura,
Scott Williams,
Tikva Hayes,
Michel Moutschen, Stefan Vasile,
Maurizio Pellecchia,
Tomas Mustelin,
Lutz Tautz
Journal of Medicinal Chemistry 11/2009; · 4.80 Impact Factor
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Shuangding Wu,
Sofie Vossius,
Souad Rahmouni,
Ana V Miletic,
Torkel Vang,
Jesus Vazquez-Rodriguez,
Fabio Cerignoli,
Yutaka Arimura,
Scott Williams,
Tikva Hayes,
Michel Moutschen, Stefan Vasile,
Maurizio Pellecchia,
Tomas Mustelin,
Lutz Tautz
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ABSTRACT: Loss of VHR phosphatase causes cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells. We recently reported that VHR is upregulated in several cervix cancer cell lines as well as in carcinomas of the uterine cervix. Here we report the development of multidentate small-molecule inhibitors of VHR that inhibit its enzymatic activity at nanomolar concentrations and exhibit antiproliferative effects on cervix cancer cells. Chemical library screening was used to identify hit compounds, which were further prioritized in profiling and kinetic experiments. SAR analysis was applied in the search for analogs with improved potency and selectivity, resulting in the discovery of novel inhibitors that are able to interact with both the phosphate-binding pocket and several distinct hydrophobic regions within VHR's active site. This multidentate binding mode was confirmed by X-ray crystallography. The inhibitors decreased the proliferation of cervix cancer cells, while growth of primary normal keratinocytes was not affected. These compounds may be a starting point to develop drugs for the treatment of cervical cancer.
Journal of Medicinal Chemistry 11/2009; 52(21):6716-23. · 4.80 Impact Factor
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Dmitri V Rozanov,
Alexei Y Savinov,
Vladislav S Golubkov,
Olga L Rozanova,
Tatiana I Postnova,
Eduard A Sergienko, Stefan Vasile,
Alexander E Aleshin,
Michele F Rega,
Maurizio Pellecchia,
Alex Y Strongin
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ABSTRACT: Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. The current suboptimal efficiency and selectivity drugs have therapeutic limitations and induce concomitant side effects. Recently, novel cancer therapies based on the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have emerged. TRAIL, a key component of the natural antitumor immune response, selectively kills many tumor cell types. Earlier studies with recombinant TRAIL, however, revealed its many shortcomings including a short half-life, off-target toxicity, and existence of TRAIL-resistant tumor cells. We improved the efficacy of recombinant TRAIL by redesigning its structure and the expression and purification procedures. The result is a highly stable leucine zipper (LZ)-TRAIL chimera that is simple to produce and purify. This chimera functions as a trimer in a manner that is similar to natural TRAIL. The formulation of the recombinant LZ-TRAIL we have developed has displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. Our results have shown that the half-life of LZ-TRAIL is improved and now exceeds 1 h in mice compared with a half-life of only minutes reported earlier for recombinant TRAIL. We have concluded that our LZ-TRAIL construct will serve as a foundation for a new generation of fully human LZ-TRAIL proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL.
Molecular Cancer Therapeutics 07/2009; 8(6):1515-25. · 5.23 Impact Factor
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Hideki Hayashi,
Michael Cuddy,
Vincent Chih-Wen Shu,
Kenneth W Yip,
Charitha Madiraju,
Paul Diaz,
Toshifumi Matsuyama,
Muneshige Kaibara,
Kohtaro Taniyama, Stefan Vasile,
Eduard Sergienko,
John C Reed
[show abstract]
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ABSTRACT: Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026;
We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS) of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy.
Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.
PLoS ONE 01/2009; 4(10):e7655. · 4.09 Impact Factor
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[show abstract]
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ABSTRACT: An oxidative enol ether rearrangement was the key methodology in the construction of steroid-spiroketal-RGD peptides. Biological studies demonstrated potent integrin CD11b/CD18 antagonistic effects.
Organic Letters 01/2009; 11(1):65-8. · 5.86 Impact Factor
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[show abstract]
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ABSTRACT: The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is caused by many disease-relevant conditions, inducing conserved signaling events collectively known as the unfolded protein response. When ER stress is excessive or prolonged, cell death (usually occurring by apoptosis) is triggered. We undertook a chemical biology approach for investigating mechanisms of ER stress-induced cell death. Using a cell-based high throughput screening assay to identify compounds that rescued a neuronal cell line from thapsigargin-induced cell death, we identified benzodiazepinones that selectively inhibit cell death caused by inducers of ER stress (thapsigargin and tunicamycin) but not by inducers of extrinsic (tumor necrosis factor) or intrinsic (mitochondrial) cell death pathways. The compounds displayed activity in several cell lines and primary cultured neurons. Mechanism of action studies revealed that these compounds inhibit ER stress-induced activation of p38 MAPK and kinases responsible for c-Jun phosphorylation. Active benzodiazepinones suppressed cell death at the level of apoptotic signal kinase-1 (ASK1) within the IRE1 pathway but without directly inhibiting the kinase activity of ASK1 or >400 other kinases tested. Rather, active compounds enhanced phosphorylation of serine 967 of ASK1, promoting ASK1 binding to 14-3-3, an event associated with suppression of ASK1 function. Reducing ASK1 protein expression using small interfering RNA also protected cells from ER stress-induced apoptosis, confirming the importance of this protein kinase. Taken together, these findings demonstrate an essential role for ASK1 in cell death induced by ER stress. The compounds identified may prove useful for revealing endogenous mechanisms that regulate inhibitory phosphorylation of ASK1.
Journal of Biological Chemistry 11/2008; 284(3):1593-603. · 4.77 Impact Factor
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Kenneth W Yip,
Paulo H C Godoi,
Dayong Zhai,
Xochella Garcia,
Jason F Cellitti,
Michael Cuddy,
Motti Gerlic,
Ya Chen,
Arnold Satterthwait, Stefan Vasile,
Eduard Sergienko,
John C Reed
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ABSTRACT: Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3-dependent manner. A 9-amino-acid peptide derived from full-length TR3 with polyarginine tail (TR3-r8) recapitulates TR3's binding specificity, displaying high affinity for Bcl-B. TR3-r8 peptide was used to screen for small molecule Bcl-B inhibitors. A fluorescence polarization assay (FPA) employing fluorescein isothiocyanate (FITC)-labeled TR3-r8 peptide (FITC-TR3-r8) and Bcl-B protein was optimized, with nonfluorescent TR3-r8 serving to demonstrate reversible, competitive binding. Approximately 50,000 compounds were screened at 3.75 mg/L, yielding 145 reproducible hits with > or =50% FITC-TR3-r8 displacement (a confirmed hit rate of 0.29%). After dose-response analyses and counterscreening with an unrelated FITC-based FPA, 6 candidate compounds remained. Nuclear magnetic resonance (NMR) showed that 2 of these compounds bound Bcl-B, but not glutathione S-transferase (GST) control protein. One Bcl-B-binding compound was unable to displace FITClabeled BH3 peptides from Bcl-B, confirming a unique binding mechanism compared with traditional antagonists of antiapoptotic Bcl-2-family proteins. This compound bound Bcl-B with Kd 1.94 +/- 0.38 microM, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrated that this compound induced Bcl-B-dependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins.
Journal of Biomolecular Screening 07/2008; 13(7):665-73. · 2.05 Impact Factor
